HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped virus classified in the Flaviviridae family. The single-strand, positive-sense viral RNA genome encodes a single polyprotein precursor that is processed into three structural proteins (C, E1, E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by host and viral proteases. The RNA genome of HCV has significant heterogeneity with six major genotypes and numerous subtypes. HCV infection is currently treated with alpha interferon (IFN)-based therapy. Although the treatment outcome is variable among the six major HCV genotypes, only 50% of treated patients infected with genotype 1 respond to therapy. The ability of HCV to establish persistent infection with great success in human has been attributed, in part, to a variety of strategies to evade host immune and IFN-induced defenses. Epidemiological studies suggest that up to 20% of acutely infected HCV patients can resolve the infection without treatment, which implies that innate and/or adaptive immune responses are indeed capable of controlling the outcome of HCV infection. The molecular mechanisms that regulate innate intracellular antiviral responses may therefore serve as pivotal points of control, potentially limiting host permissiveness for HCV replication and favorably modulating subsequent adaptive immune responses. A major consequence of virus infection is the induction of the type I interferons (IFN) including IFN alpha and beta, which are cytokines of immune system with essential function in innate and adaptive immune response. Secreted IFN then triggers hundreds of IFN stimulated genes (ISGs) expression which establishes an antiviral state in the surrounding of virus-infected cells to limit viral replication, spread and infection. Double-stranded RNA (dsRNA) is the replication intermediate of RNA virus and is produced during viral replication. It has long been thought that dsRNA is a marker of virus infection because its molecular characteristic seems to allow for the distinction of self and nonself RNA. Poly(I:C), which is synthetic dsRNA analogue, has been used widely as an alternative to viral dsRNA for studying innate immunity mechanism in the cells because of its capability of activation of IFN. Interferon alpha or beta production is usually measured by using ELISA or real-time polymerase chain reaction methods. The disadvantage of these methods is either time-consuming or insensitive. We have developed a novel reporter assay, IFN-beta-SEAP, which secreted alkaline phosphatase (SEAP) is under the control of human IFN beta promoter sequence. It is designed for monitoring human IFN promoter activation by detection of SEAP activity in the culture media. We have demonstrated that SEAP activity in the media of cell culture is specifically induced by poly(I:C). This assay is about 40 to 150-fold more sensitive than IFN-Luc reporter assay in the cells we tested. Thus, such reporter assay will be very useful for studying innate immune responses to various stimuli because of its high sensitivity and convenience to time-course analysis. Using this IFN-beta-SEAP reporter assay and small interfering RNA approach, we have characterized several genes which exert profound effect on activation of IFN-beta gene in host after stimulation with HCV RNA or poly (I:C). Also, we have successfully identified and characterized several cell lines with specific knock-down genes related to innate immunity. Currently, we start to assess the infectivity of HCV patient serum samples on innate immunity knock-down cell lines.